Studies On The Preparation And Biological Functions Of Antioxidant Peptides From Crocodile Tenderloin

In this study,antioxidant peptides were prepared from crocodile tenderloin,a by-product in crocodile processing industry,through enzymatic hydrolysis.And the antioxidant activities of antioxidant peptides from crocodile tenderloin(CTAP)were evaluated from three aspects:antioxidant activity in vitro,antioxidant damage repair function in vivo and antioxidant stability.Several isolation and separation technologies were done on CTAP and LC-MS/MS was used to identify their amino acid sequences.Apart from that,immunomodulatory function,cytotoxicity and effects on the mouse growth and organ parameters of the antioxidant peptides were also studied.The main research work and findings were summarized as follows:Based on the single factor experiment with DPPH·free radical scavenging rate and free amino acid content as indicators,Response Surface Methodology(RSM)was used to optimize the processing conditions of hydrolysis of CTAP,and the results were:for papain,enzyme-substrate ratio was 3%,enzymatic temperature was 55?,hydrolytic time was 4 h and substrate concentration was 5.5%.The biochemical properties of CTAP were measured and analyzed by various methods.Results showed that the powder of CTAP exhibited irregular flaky structure and rough surface.They contained high protein(72.64%),low sugar(6.32%),various metal elements beneficial to human body and abundant amino acids.CTAP also had a wide range of molecular mass,which almost less than 3 kDa.Four different chemical models in vitro were used to evaluate the antioxidant activities of CTAP Results showed that their scavenging activities for DPPH radicals,ABTS+·radicals,·OH radicals activities and reducing power were strong.HaCaT cells induced by H2O2 oxidative damage were used as models to show that CTAP had repair function.The effects of temperature,pH,metal ions,food ingredients,UV,freezing and thawing and simulated gastrointestinal digestion in vitro on the ABTS+·radical scavenging activity of CTAP were investigated to determine their stability.Results showed that:CTAP had good stabilities of temperature;they were stable in neutral and alkaline conditions,but lost their antioxidant activities in acid condition;The Al3+,Cu2+and Fe3+ at an increasing concentration significantly decreased the antioxidant activity of them,while K+,Ca2+,Mg2+ and 2Zn2+ had no effect;And food ingredients,UV irradiation,repeated freezing and thawing and mimic gastrointestinal digestion in vitro had no significant difference on their antioxidant activity.CTAP were isolated and purified by membrane ultrafiltration and consecutive chromatographic methods including ion-exchange chromatography,gel filtration chromatography and RP-HPLC.And the amino acid sequences of the purified CTAP were identified using LC-MS/MS;Proliferation and phagocytic activity experiments indicated that CTAP had a certain immunomodulatory effect on Raw 264.7 cells.The effect of detection of L02 cell proliferation by MTT assay showed that CTAP were not cytotoxic.Mice experiment showed that CTAP had tenderloin no significant effect on the body weight and organ coefficient of mice,and they did not cause obvious pathological damage to the five internal organs of mice.In this paper,the optimal enzymatic hydrolysis conditions,biochemical properties,antioxidant activities and other biological functions of CTAP were studied.In the mean while,various methods were used to separate and purify the antioxidant activity components of the CTAP.Not only can it increase the added value of crocodile products,but also reduce environmental pollution,and have a certain significance for the full use of crocodile resources.